Biosynthesis and Molecular Genetics of Clavulanic Acid

The biosynthesis of clavulanic acid and related clavam metabolites is only now being elucidated. Understanding of this pathway has resulted from a combination of both biochemical studies of purified biosynthetic enzymes, and molecular genetic studies of the genes encoding these enzymes. Clavulanic acid biosynthesis has been most thoroughly investigated in Streptomyces clavuligerus where the biosynthetic gene cluster resides immediately adjacent to the cluster of cephamycin biosynthetic genes. A minimum of eight structural genes have been implicated in clavulanic acid biosynthesis, although more are probably involved. While details of the early and late steps of the pathway remain unclear, synthesis proceeds from arginine and pyruvate, as the most likely primary metabolic precursors, through the monocyclic -lactam intermediate, proclavaminic acid, to the bicyclic intermediate, clavaminic acid, which is a branch point leading either to clavulanic acid or the other clavams. Conversion of clavaminic acid to clavulanic acid requires side chain modfication as well as inversion of ring stereochemistry. This stereochemical change occurs coincident with acquisition of the -lactamase inhibitory activity which gives clavulanic acid its therapeutic and commercial importance. In contrast, the other clavam metabolites all arise from clavaminic acid with retention of configuration and lack -lactamase inhibitory activity.     

A class II patatin promoter is under developmental control in both transgenic potato and tobacco plants

Summary A new member of the patatin gene family belonging to the class II subfamily was isolated and characterized by DNA sequencing. In order to study the expression profile of this gene, the promoter was fused to the -glucuronidase gene and transferred to potato and tobacco. Histochemical analysis revealed high expression in a few defined cells in potato tubers and in a specific layer of both potato and tobacco root tips. In contrast to the developmentally and metabolically regulated class I patatin gene B33 this gene was not inducible by elevated levels of sucrose. Expression of this chimaeric gene was also found in callus and suspension cultures of potato.     

Development and isoproterenol-induced regulation of adrenoceptor binding in cultured rat neocortical explants is seen only with the beta-1, not with the beta-2 subtype

The presence and time-course of -adrenoceptor density in cultured explants of neocortex obtained from 6-day-old rat pups were investigated using a [¹²⁵I]ICYP binding assay. A delayed, but more pronounced, increase in the receptor expression was observed as compared to the situation previously described in vivo. These changes only occurred for the ₁-subtype of the receptor, whereas the ₂-subtype binding remained constant up to 3 weeks in vitro. The delay of ₁-adrenoceptor expression may be due to the incomplete presence of the proper maturational input, and the late enhancement of receptor expression to upregulation related to the absence in vitro of noradrenergic input. Decreased -adrenoceptor levels could be induced by chronic treatment of the -agonist isoproterenol (1 M) introduced either for 3 or 13 days. Again, changes in density were found only for the ₁-adrenoceptor binding sites. There is no reduction of receptor density following return to control conditions for 10 days after a 3-day treatment with isoproterenol, demonstrating the ability of this model to attain its final receptor density notwithstanding the developmental insult.Special issue dedicated to Dr. Robert Balázs     

The use of norbornene derivatives in the synthesis of conformationally constrained peptides and pseudo-peptides

The desymmetrisation of endo-norborn-5-ene-2,3-dicarboxylic anhydride by proline esters has been used to prepare conformationally constrained pseudo-peptides with two peptide chains parallel to one another. A Curtius rearrangement on the desymmetrisation adduct produced the corresponding isocyanate which was used to prepare both a peptide incorporating an endo-2-amino-3-carboxy-norborn-5-ene unit, and a pseudo-peptide with two peptide chains parallel to one another but offset by the presence of a urea unit. The conformational analysis of the resulting peptides was carried out, and the norbornene unit was found to induce the formation of -turns and parallel -sheets.     

Modulation of the serotonin-activated K+ channel by G protein subunits and nucleotides in rat hippocampal neurons

In hippocampal neurons, 5-hydroxytryptamine (5-HT) activates an inwardly rectifying K⁺ current via G protein. We identified the K⁺ channel activated by 5-HT (K_5-HT channel) and studied the effects of G protein subunits and nucleotides on the K⁺ channel kinetics in adult rat hippocampal neurons. In inside-out patches with 10 m 5-HT in the pipette, application of GTP (100 m) to the cytoplasmic side of the membrane activated an inwardly rectifying K⁺ channel with a slope conductance of 36±1 pS (symmetrical 140 mm K⁺) at –60 mV and a mean open time of 1.1±0.1 msec (n=5). Transducin activated the (K_5-HT) channels and this was reversed by -GDP. Whether the K_5-HT channel was activated endogenously (GTP, GTPS) or exogenously (), the presence of 1 mm ATP resulted in a 4-fold increase in channel activity due in large part to the prolongation of the open time duration. These effects of ATP were irreversible and not mimicked by AMPPMP, suggesting that phosphorylation might be involved. However, inhibitors of protein kinases A and C (H-7, staurosporine) and tyrosine kinase (tyrphostin 25) failed to block the effect of ATP. These results show that G activates the G protein-gated K⁺ channel in hippocampal neurons, and that ATP modifies the gating kinetics of the channel, resulting in increased open probability via as yet unknown pathways.     

cDNA cloning and expression of a potato (Solanum tuberosum) invertase

A cDNA clone encoding an invertase isoenzyme has been isolated from a potato leaf cDNA library. The deduced amino acid sequence shows significant similarities to previously characterised invertases. The highest degree of overall similarity, including the signal peptide sequence, is to carrot cell wall invertase, suggesting that the potato gene encodes an apoplastic enzyme. Expression of the gene, as determined by RT-PCR, is detected in stem and leaf tissue, and at lower levels in tuber, but is absent from roots.     

Enzymatic syntheses of GlcNAcβ1-2Man and Galβ1-4GlcNAcβ1-2Man as components of complex type sugar chains

GlcNAc1-2Man and GlcNAc1-6Man were synthesized using the reverse hydrolysis activity of -N-acetylglucosaminidase from both jack beans and Bacillus circulans. In turn, Gal1-4GlcNAc1-2Man and Gal1-4GlcNAc1-6Man were synthesized regioselectively using the transglycosylation activity of -galactosidase from Diplococcus pneumoniae and B. circulans, respectively. These di- and trisaccharides are important components of complex type sugar chains and will be used as intermediates in our synthetic studies. Abbreviations: pNp--GlcNAc, p-nitrophenyl 2-acetamido-2-deoxy--D-glucopyranoside; pNp--Gal, p-nitrophenyl -D-galacto-pyranoside     

Site-specific oligodeoxynucleotide binding to maize Adh1 gene promoter represses Adh1-GUS gene expression in vivo

There is a 36 bp tract of extreme homopurine/homopyrimidine (PuPy) asymmetry in the maize Adh1 gene promoter (from –44 to –79) that is S1-hypersensitive in plasmids under supercoil tension. Oligodeoxynucleotides corresponding to the PuPy tract were designed to examine the secondary structure of the region and address the possible role of the tract in gene regulation. On the basis of oligodeoxynucleotide band-shift and DNase I footprinting analyses, it was concluded that the homopyrimidine oligodeoxynucleotide can form a triple helix with the duplex PuPy tract in vitro. Transient assays in protoplasts, suspension cells, and seedling roots show that the homopyrimidine oligodeoxynucleotide is also capable of repressing Adh1-GUS gene expression during co-transformation, presumably by the formation of a triple helix with the PuPy tract in vivo. The complementary homopurine oligodeoxynucleotide would not form a triple helix in vitro, nor would it repress Adh1-GUS in vivo. We propose that triple helix formation is a potential regulatory phenomenon in vivo, and that an intraregion triple helix could occur within the Adh1 promoter via the formation of H-DNA.     

Interactions between esterified whey proteins (alpha-lactalbumin and beta-lactoglobulin) and DNA studied by differential spectroscopy

Spectroscopic study of interactions between esterified whey proteins and nucleic acids, at neutral pH, showed positive differential spectra over a range of wavelength between 210 and 340 nm. In contrast, native forms of whey proteins added to DNA did not produce any differential spectra. The positive difference in UV absorption was observed after addition of amounts of proteins as low as 138 molar ratio (MR) of protein/DNA, indicating high sensitivity of the applied method to detect interactions between basic proteins and DNA. UV-absorption differences increased with MR of added whey protein up to saturation. The saturation points were reached at relatively lower MR in the case of methylated forms of the esterified protein as compared to its ethylated form. Saturation of nucleic acid (2996 bp long) was achieved using 850 and 1100 MR of methylated -lactoglobulin and of methylated -lactalbumin, respectively. Saturation with ethylated forms of the proteins was reached at MR of 3160 and 2750. Lysozyme, a native basic protein, showed a behavior similar to what was observed in the case of methylated forms of the dairy proteins studied. However, in the case of lysozyme, saturation was achieved at relatively lower MR (700). Methylated -casein failed to give positive spectra at pH 7 in the presence of DNA. It interacted with DNA only when the pH of the medium was lowered to 6.5, below its pI. Generally, amounts of proteins needed to saturate nucleic acid were much higher than those needed to neutralize it only electrostatically, demonstrating the presence on DNA of protein-binding sites other than the negative charges on the sugar-phosphate DNA backbones. Addition of 0.1% SDS to the medium suppressed totally all spectral differences between 210–340 nm. The presence of 5 M urea in the medium reduced only the spectral differences between 210–340 nm, pointing to the role played by hydrophobic interactions. Peptic hydrolysates of esterified and native proteins or their cationic fractions (pH > 7) produced negative differential spectra when mixed with DNA. The negative differences in UV absorption spectra were the most important in the case of peptic hydrolysates of methylated derivatives of whey proteins.     

Expression of an auxin-inducible promoter of tobacco in Arabidopsis thaliana

The expression of the auxin-inducible Nt103-1 gene of tobacco was studied in Arabidopsis thaliana. For this purpose we introduced a gene fusion between the promoter of the gene and the -glucuronidase reporter gene (GUS) into Arabidopsis thaliana. The expression and location of GUS activity were studied histochemically in time and after incubation of seedlings on medium containing auxins or other compounds. The auxins 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), and 1-naphthylacetic acid (1-NAA) were able to induce GUS activity in the root tips of transgenic seedlings. The auxin transport inhibitor 2,3,5-triiodobenzoic acid was able to induce GUS activity not only in the root tip, but also in other parts of the root. Induction by the inactive auxin analog 3,5-dichlorophenoxyacetic acid was much weaker. Compounds like glutathione and the heavy metal CuSO₄ were weak inducers. GUS activity observed after induction by glutathione was located in the transition zone. Salicylic acid and compounds increasing the concentration of hydrogen peroxide in the cell were also very well able to induce GUS activity in the roots. The possible involvement of hydrogen peroxide as a second messenger in the pathway leading to the induction of the Nt103-1 promoter is discussed.     

The response of the antioxidant defense system in rat hepatocytes challenged with oxysterols is modified by Covi-ox

Suspension cultures of isolated rat hepatocytes were used to investigate whether 7-ketocholesterol and cholestane-3,5,6-triol exert oxidative stress in cells as manifested by increased lipid peroxidation and the induction of the antioxidant enzymes catalase, glutathione peroxidase and superoxide dismutase. The oxysterols were found to increase the levels of both superoxide dismutase and catalase and to have variable effects on glutathione peroxidase activity. Increased lipid peroxidation was not observed, indicating that the endogenous antioxidant defense system was capable of protecting against any oxidative stress that might otherwise by exerted by 7-ketocholesterol or cholestane-3,5,6-triol. Covi-ox, a natural tocopherol blend reduced the effects of both oxysterols on the antioxidant enzymes. A concurrent reduction in the production of thiobarbituric acid-reactive substances in Covi-ox-treated cells is indirect evidence that reactive oxygen species were produced by oxysterols in hepatocyte suspension cultures.     

Tail-to-tail orientation of the Atlantic salmon alpha- and beta-globin genes

We report the cloning of a cDNA and two corresponding -globin genes of the Atlantic salmon (Salmo salar L.) as well as two genes for -globins. Nucleotide sequence analysis of the cDNA shows that the predicted -globin peptide comprises 148 amino acids with a calculated molecular mass of 16,127 Da and an overall amino acid similarity of 40–50% to higher vertebrates and 60–90% to fish sequences. The study of the genomic organization of - and -globin genes shows that, as is the case in Xenopus, the salmon genes are adjacent. Two sets of linked - and -globin genes were isolated and restriction-enzyme polymorphisms indicate that they belong to two distinct loci, possibly as a result of the salmon tetraploidy. In each locus the - and -globin genes are oriented 3 to 3 relative to each other with the RNA coding sequences located on opposite DNA strands. This is the first evidence for this type of arrangement found for globin genes. Moreover, while the linkage found in salmon and Xenopus supports the hypothesis of an initial tandem duplication of a globin ancestor gene, our results raise the question of the actual original orientation of the duplicated genes. Correspondence to: F. Gannon